Journal of Medicinal Plants Research Vol. 6(40), pp.

5311-5316, 17 October, 2012

Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1352
ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Isolation of mangiferin and amyloid -protein from nhexane extract of roots of Wrightia tomentosa
Kandasamy Nagarajan1, Ira Sharma1*, Surendra Kumar Srivastava2, Ramesh B. Bodla3,
Anjali malik2, Aanchal rathi1 and U. K. Bajaj1
1

Department of Pharmaceutical Chemistry, KIET School of Pharmacy, Ghaziabad, India.

2
IIMT College of Medical Sciences, Meerut, India.
3
Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), New Delhi, India.
Accepted 11 April, 2012

The aim of this study is to identify and characterize the bioactive principles from the roots of Wrightia
tomentosa. For isolation, the compound, the dried root powder of W. tomentosa was subjected to hot
extraction with n-hexane to chromatography. Two compounds (WTRHF4 and WTRHF7) were isolated
and purified by chloroform: toluene (7.5: 2.5) and chloroform: ethyl acetate (6: 4), respectively. The
infra-red (I.R) spectra of WTRHF4 showed specific absorption bands for proteins, viz., 3689 to 3629 cm-1
for COOH stretching; 2375 cm-1 for NH3+ stretching; 1726 cm-1 for COO- stretching; and the Mass
spectrum of WTRHF4 showed the parent molecular ion (M )+ cum base peak at m/z 663.5. In addition, the
I.R spectra of WTRHF7 showed specific absorption bands for flavonoids, viz., 3780 cm-1 for stretching
alcohol and phenol; 1098 to 1048 cm-1 for C-O-C stretching; 1445 to 1375 cm-1 for O-H ip phenol; 1739
cm-1 for C=O stretching and the mass spectra of WTRHF7 showed a parent molecular ion (M+1)+ peak at
m/z 423.4 which corresponds to the molecular formula C19H18O11. From the physical, chemical and
spectral characteristics, WTRHF4 and WTRHF7 were concluded as amyloid -protein (A17-28) and
mangiferin, respectively.
Key words: Wrightia tomentosa, n-hexane, roots, mangiferin, amyloid -protein.
INTRODUCTION
Natural products have been one of the most successful
sources of medicines. Each plants is like a chemical
factory capable of synthesizing unlimited number of
highly complex and unusual chemical substances whose
structures could otherwise escape the imagination
forever (Shinde and Dhalwal, 2007)
Wrightia tomentosa Roem and Schult, family
Apocynaceae, is widely distributed at an altitude of 600m
in the Himalayas. A novel isoflavone, wrightiadione
isolated from the plant possess cytotoxic activity against
murine P388 lymphocytic leukaemia cell line (Lee et al.,
1992). The root barks are found to be useful in snake bite
and scorpion-stings (Kirtikar and Basu, 1980). However,
the ethanolic bark and leaf extract of W. tomentosa

*Corresponding author. E-mail: sharma_26_1988@yahoo.com.

Tel: +919457818063.

possesses significant anti-allodynic effects with no

observable signs of toxicity and antihyperglycemic activity
in streptozotocin induced diabetic rats. The alcoholic
extract of W. tomentosa dried bark was reported to
exhibit markedly high antioxidant potency (IC50 value of
75.0 g/ml from DPPH radicals scavenging assay),
suitable for prevention of human disease. The butanol
extract of the plant was shown to have anti-microbial
activity against both gram positive and gram negative
organisms. The leaf extract of W. tomentosa has proved
to be extremely useful against non-tuberculoses
mycobacterium (NTM) infections (Nagarajan, 2008),
which are becoming a major concern for hospitals and
medical clinics. W. tomentosa leaf and fruit showed the
best antibacterial activity among the various extract used,
methanol extract followed by ethyl acetate extracts
showed better antibacterial activity (Kaneria et al., 2009).
The methanol (dichloromethane) extract of the leaves
of W. tomentosa was tested for anti-inflammatory activity

J. Med. Plants Res.

T (%)

5312

cm

-1

Figure 1. Slice through the graph representing I.R spectra of bioactive leads WTRHF4 from W. tomentosa.

by dextran induced paw oedema rat model and it was

found that the extract significantly ameliorate the dextran
induced oedma (David, 2010).
Since the root portion has not been extracted and
isolated for its active constituents in solvent like ethanol,
this is the first attempt that the authors planned to carry
out on this. Our objective is to study a design of isolation
and characterization of bioactive pure components from
the roots of W. tomentosa.
MATERIALS AND METHODS
Plant materials
The roots of W. tomentosa were collected from the hills of yercavd
forest, Salem district of Tamilnadu and identified by the method of
Mattehew (1982), and authenticated by an acknowledged botanist,
Professor M.B. Viswanathan, Co-ordinator, Centre for Herbal Drug
Discovery and Development of the Research Department of
Bharathidasan University, Tiruchirappalli, Tamilnadu, India, and the
voucher specimen was thereafter deposited at Bharathidasan

University (BDUT/545).
Extraction and isolation
The roots of W. tomentosa were dried at room temperature and
reduced to a coarse powder. The powder material was subjected to
qualitative tests (Harborne, 1998) for the identification of various
phytoconsitituents like alkaloids, saponins and proteins. Then the
powder (180 g) was subjected to Soxhlet extraction with n-hexane
separately for 72 h at a temperature of 69C. The extracts were
concentrated and the solvent was completely removed by rotary
vacuum evaporator (Buchi) then light green residue was obtained.
The concentrated n-hexane root extract (1 g) were taken in a
china dish separately and heated continuously on a water bath by
gradually adding n-hexane in a small portion with constant stirring
till desired consistency was obtained. Silica gel (for column
chromatography, 200 mesh size) was then added (weighed quantity
20 g for root extract) slowly with continuous mixing with steel
spatula till desired consistency of the mixture was obtain. It was air
dried and larger lumps were broken to get a smooth free flowing
mixture.
A column of 5.0 feet length and 16 mm of internal diameter was
taken and dried. The lower end of the column was plugged with

5313

T (%)

Nagarajan et al.

cm

-1

Figure 2. Slice through the graph representing I.R spectra of bioactive leads WTRHF7 from W. tomentosa.

absorbent cotton. The column was clamped and fitted in vertical

position on a stand. The column was then half-filled with n-hexane;
silica gel was then poured in small portions and allowed to settle
gently until the necessary length of the column was obtained. The
dried silica gel slurry containing the n-hexane extract of root was
poured in the column separately and then eluted successively with
different solvents in the order of toluene [toluene: chloroform (7.5:
2.5), toluene: chloroform (5: 5), toluene: chloroform (2.5: 7.5)];
chloroform [chloroform: ethyl acetate (8: 2), chloroform: ethyl
acetate (6: 4), chloroform: ethyl acetate (4: 6), chloroform: ethyl
acetate (2: 8)]; ethyl acetate [ethyl acetate: ethanol: water (100:
13.5: 10), ethyl acetate: ethanol (7.5: 2.5), ethyl acetate: ethanol (
5: 5), ethyl acetate: ethanol ( 2.5: 7.5)]; ethanol [chloroform:
ethanol: glacial acetic acid (9: 9: 1)]; n-butanol [n-butanol: water(6:
4), n-butanol: water (2: 8), and n-butanol: acetic acid: water (4: 4:
1)]. Twenty fractions were collected in a conical flask and marked.
The marked fractions were subjected to TLC to check homogeneity
of various fractions (Stahl, 1969). Chromatographically, identical
various fractions (having same Rf values) were combined together

and concentrated. They were then crystallized with suitable solvent

systems.
Physico-chemical characterization of pure isolate
Two pure bioactive leads were isolated and the isolated pure
fractions were tentatively identified by qualitative chemical analysis.
Further identification and characterization was done using I.R and
mass spectra analysis.
Infra red (I.R) spectra analysis
The I.R spectra were recorded on Perkin Elmer RX1 at
Sophisticated Analytical Instrument Facility (SAIF) Central Drug
Research Institute, Lucknow. The isolated test compounds were
subjected to I.R using KBr/chloroform with the study of absorption
of infra-red radiation functional groups clearly (Silverstein and

5314

J. Med. Plants Res.

Figure 3. Mass spectrum of a protein component WTRHF4 of W. tomentosa.

Webster, 2005). The I.R results in the two isolated leads are
expressed subsequently in Figure 1 and Figure 2 with the work.

presence of protein for WTRHF4 and flavonoids for

WTRHF7 as major active constituents in addition to the
presence of alkaloids, saponins, steroids, and glycosides.

Mass spectra analysis

The electrospray mass spectral for the isolated two test compounds
were recorded as indicated in Figures 3 and 4 on Thermo Finnigan
LCQ advantage max ion trap mass spectrometer at SAIF, CDRI,
and Lucknow. The 10 l samples (dissolved in solvent such as
methanol/acetonitrile/water) were introduced into the ESI source
through Finnigan Surveyor Autosampler. The mobile phase (90: 10
MeOH/ACN: H2 O) was selected based on the preliminary
investigation such as solubility and it was maintained at the flow
rate of 250 l/min by MS pump. Ion spray voltage was set at 5.3 KV
and capillary voltage 34 V (Gross, 2004). The MS scan run up to
2.5 min and the spectra print outs was arranged for over 10 scan at
peak top in TLC. The mass spectra give information on various
types of peaks and determining the molecular formula for the
isolated compounds after successful interpretation.
The mass spectral data of WTRHF7 as from Figure 5, showed
various significant peaks in the spectrum. When analyzing the
spectrum of narrow region 200 to 600 m/z, we found that the
characteristic peak was obtained at 423.4 m/z which was confirmed
as (M+1 ) peak for the component magniferin, as further observed
with preliminary data results, through I.R and C,H,N analysis.

RESULTS AND DISCUSSION

Preliminary phytochemical screening
The results of phytochemical screening showed the

Isolation and preliminary identification of bioactive

leads from W. tomentosa roots
Elution of root drug in column with 25% toluenechloroform, that is, fraction 4 yielded dark green
amorphous powder, Rf: 0.90 (ethyl acetate: n-hexane; 6:
4) and positive with Biuret reagent for proteins and was
designated as WTRHF4.
Similarly, the elution of root drug in column with 60%
chloroform- ethyl acetate, that is, fraction 7, yielded
brown colour powder Rf : 0.83 (ethyl acetate: n-hexane;
6: 4) and positive with Shinoda test for flavonoid and was
subsequently designated as WTRHF7. The two bioactive
leads (WTRHF4 and WTRHF7) were identified as protein
and flavonoids by TLC analysis and qualitative chemical
analysis.
Physico-chemical characterization of bioactive leads
from W. tomentosa
The isolated bioactive leads, WTRHF4 and WTRHF7 were
characterized by I.R and mass spectral data. Preliminary
phytochemical screening and TLC results revealed that
the pure components (WTRHF4 and WTRHF7) were

Nagarajan et al.

5315

Figure 4. Mass spectrum of isoflavonoid component WTRHF7 of W. tomentosa.

Figure 5. Chemical structure of mangiferin.

basically protein and flavonoids class compound.

Test compound (WTRHF4)
The I.R spectra shows characteristic functional group

bands for the presence of amyloid -protein at 3689 to

3629 cm-1 (COO-H stretching carboxylic acid, O-H
-1
stretching alcohols and phenols); 3411 cm ( N-H
-1
stretching pyrrole); 2924 cm (NH stretching H bended
Pyrrole); 2375cm-1 (NH+3 stretching primary ammonium/
NH+2 stretching primary amine/ NH+ stretching secondary

5316

J. Med. Plants Res.

-1

amine); 2153 cm (-NC- isonitrile / -N N stretching

-1
diazo compounds); 1726 cm (COO stretching C=O
stretching aldehyde/ ketones/- keto ester/ amide
carbamate); 1594 to 1441 cm-1 (C-H stretching five ring
heteroaromatic ring skeleton); 1373 cm-1 (O-H ip
alcohol/phenol); 1219 cm-1 (aromatic C-H and aromatic
hydrocarbon); 1085 cm-1 (C-O-C stretching furan); 769
-1
-1
cm (COO formates); and 673 cm (COO
benzoate). Mass spectral analysis also indicates the
evidence of amyloid -protein (A 17- 28) as the final
structure of isolated protein with the corresponding
+
molecular (M ) ion cum base peak at 663.5 m/z. All these
spectral data, suggests that the protein root isolate
(WTRHF4) eluted from column was found to be amyloid
-protein (A 17-28).
Test compound (WTRHF7)
The I.R spectra shows characteristic functional groups
-1
bands for the presence of mangiferin at 3780 cm (OH
-1
stretching alcohol and phenol); 3022cm (Aromatic C-H
stretching aromatic hydrocarbon); 2987 to 2940 cm-1 (CH stretching alkanes); 2090 to 1890 cm-1 (overtones of
heteroaromatic compounds); 1739 cm-1 (C=C stretching
cyclic alkenes; C=O stretching aldehyde); 1445 to 1375
cm-1 (O-H ip alcohols and phenols =CH ip alkenes);
1098 to 1048 cm-1 (C-O-C stretching); and 937 to 848 cm1
(CH ), Mass spectral analysis also indicates the
evidence of mangiferin as the final structure of isolated
flavonoid class with the corresponding (M+1)+ ion peak at
m/z 423.4 which corresponds to the molecular formula
C19H18O11 as observed from Figure 5. All these spectral
data suggests that the flavonoid root isolate WTRHF7
eluted from the column was found to be mangiferin.
Hence, from the physical, chemical and spectral
characteristics, WTRHF4 and WTRHF7 were concluded
as amyloid -protein (A17-28) and mangiferin,
respectively.
ACKNOWLEDGEMENTS
The authors are very much thankful to Dr. D.K Dikshit,
Deputy Director and Head, Sophisticated Analytical
Instrumental Facility (SAIF), Central Drug Research
Institute (CDRI), Lucknow for providing all spectral data in
time. We also remain thankful to Dr. Vijender singh,
Principal, KIET School of Pharmacy for his Co-operation
and moral support.

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